Endoglin As an Adhesion Molecule in Mature and Progenitor Endothelial Cells: a Function Beyond TGF-Β

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MINI REVIEW published: 30 January 2019 doi: 10.3389/fmed.2019.00010

Endoglin as an Adhesion Molecule in Mature and Progenitor Endothelial Cells: A Function Beyond TGF-β

Elisa Rossi 1,2*, Carmelo Bernabeu 3 and David M. Smadja 1,2,4,5

1 Université Paris Descartes, Sorbonne Paris Cité, Paris, France, 2 Inserm UMR-S1140, Paris, France, 3 Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientíﬁcas, Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain, 4 Department of Hematology, AP-HP, Hôpital Européen Georges Pompidou, Paris, France, 5 Laboratory of Biosurgical Research, Carpentier Foundation, Hôpital Européen Georges Pompidou, Paris, France

Endoglin (ENG) is a transmembrane glycoprotein expressed on endothelial cells that functions as a co-receptor for several ligands of the transforming growth factor beta (TGF-β) family. ENG is also a recognized marker of angiogenesis and mutations in the endoglin gene are responsible for Hereditary Hemorrhagic Telangiectasia (HHT) type 1, a vascular disease characterized by defective angiogenesis, arteriovenous malformations,

Edited by: telangiectasia, and epistaxis. In addition to its involvement in the TGF-β family signaling Peter S. Steyger, pathways, several lines of evidence suggest that the extracellular domain of ENG has Oregon Health and Science University, a role in integrin-mediated cell adhesion via its RGD motif. Indeed, we have described United States a role for endothelial ENG in leukocyte trafﬁcking and extravasation via its binding to Reviewed by: Eleni Papakonstantinou, leukocyte integrins. We have also found that ENG is involved in vasculogenic properties of Aristotle University of Thessaloniki, endothelial progenitor cells known as endothelial colony forming cells (ECFCs). Moreover, Greece Valerie Kouskoff, the binding of endothelial ENG to platelet integrins regulate the resistance to shear during University of Manchester, platelet-endothelium interactions under inﬂammatory conditions. Because of the need for United Kingdom more effective treatments in HHT and the involvement of ENG in angiogenesis, current Michelle Letarte, Hospital for Sick Children, Canada studies are aimed at identifying novel biological functions of ENG which could serve as *Correspondence: a therapeutic target. This review focuses on the interaction between ENG and integrins Elisa Rossi with the aim to better understand the role of this protein in blood vessel formation driven [email protected] by progenitor and mature endothelial cells.

Specialty section: Keywords: endoglin, endothelial progenitors, EPC, ECFCs, HHT1, integrins, TGF-β This article was submitted to Translational Medicine, a section of the journal Frontiers in Medicine ENDOGLIN STRUCTURE AND FUNCTION Received: 27 June 2018 Endoglin (ENG; also known as CD105) is a type I transmembrane glycoprotein predominantly Accepted: 14 January 2019 expressed in endothelial cells (ECs) (1, 2) and endothelial colony-forming cells (ECFCs) Published: 30 January 2019 (3). It is known as an essential co-receptor for the transforming growth factor β (TGF- Citation: β) family, playing an important role in angiogenesis. At the cell surface, ENG associates Rossi E, Bernabeu C and Smadja DM with TGF-β type I receptors, including the ECs-specific ALK1 (activin receptor-like kinase (2019) Endoglin as an Adhesion β Molecule in Mature and Progenitor 1) and the ubiquitous ALK5, as well as to a TGF- type II receptor. In this receptor Endothelial Cells: A Function Beyond complex, ENG and ALK1 are able to bind to bone morphogenetic protein 9 (BMP9; TGF-β. Front. Med. 6:10. also known as growth differentiation factor 2 [GDF2]), a member of the TGF-β family, doi: 10.3389/fmed.2019.00010 with much higher affinity than to TGF-β1 and mediate its proliferation signal (4–7).

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Upon ligand binding, the TGF-β signaling receptor complex VASCULAR PATHOPHYSIOLOGY OF phosphorylates members of the Smad family of transcription ENDOGLIN IN HEREDITARY HEMORRAGIC factors which, in turn, undergo nuclear translocation to regulate TELANGECTASIA (HHT) gene expression (Figure 1A). In addition to the membrane bound form of endoglin, Hereditary Hemorrhagic Telangiectasia (HHT) is an a circulating form of the ENG ectodomain, was found to autosomal dominant disease with a prevalence of one case be released upon a C-terminal cleavage of ENG by matrix per 5,000–8,000 individuals (25, 26). HHT patients develop metalloproteinase 14 (MMP-14) (8, 9). Increased levels of muco-cutaneous lesions known as telangiectasia in the nose, circulating endoglin have been detected at early stages of mouth, and gastrointestinal tract, as well as larger AVMs preeclampsia (5, 10) in hypertensive or diabetic patients (11) in major organs such as the lung, liver, and brain (25). The and in certain cancer patients (12), suggesting its role as telangiectasia are comprised of fragile vessels that are susceptible a predictive biomarker in these pathologies. Interestingly, to rupture and hemorrhage, which means that HHT patients can a detailed characterization of plasma from women with suffer recurrent anemia following frequent and severe bleeding preeclampsia has revealed that circulating endoglin can be episodes. Mutations in the endoglin gene (ENG) are responsible complexed within exosomes, rather than as individual soluble for HHT type 1 (HHT1) (27), while mutations in activing endoglin (13). receptor-like kinase (ACVRL1 alias ALK1) are responsible for A pioneer article on human ENG described that this HHT2 (2, 27). Taken together, HHT1 and HHT2 account for glycoprotein is present on ECs as a dimer (two subunits more than 80% of all HHT cases, whereas a small number of of ∼90-kDa), each displaying the tripeptide arginine-glycine- patients show mutations in SMAD4 (MADH4) or in BMP9 aspartic acid (RGD) in the extracellular region of the protein gene (GDF2), which are responsible for less common HHT (1). The RGD motif and homologous sequences in mouse variants such as a combined syndrome with juvenile polyposis and pig are recognition motifs for cell surface integrins (JP-HHT) and HHT5, respectively (28, 29). Remarkably, all present in extracellular matrix proteins such as fibronectin, of these genes code for proteins involved in the TGF-β family tenascin, thrombospondin, vitronectin, von Willebrand factor signaling pathways (30). Nowadays, the most widely accepted as well as fibrinogen and prothrombin (14–16). Integrin- hypotheses for AVMs formation in HHT1 and HHT2 involves mediated adhesion is involved in hemostasis, thrombosis, and a “first hit” (gene haploinsufficiency) based on the loss of inflammation, processes in which the endothelium plays a one functional allele Eng+/− (HHT1) or ALK1+/− (HHT2), critical role. Therefore, the presence of the RGD motif within which causes a 50% reduction in protein expression, followed a hydrophilic environment (1) and its accessibility in the 3D by a “second hit” based on an angiogenic trigger such as structure of ENG (7), clearly suggest the involvement of ENG inflammation, hypoxia or vascular injury (31). Together, these in integrin binding, as postulated in early studies (17, 18). In events may induce a deficient endothelial function of ENG fact, endothelial endoglin binds to leukocyte integrins, allowing that could result in HHT vascular lesions. The relevant role leukocyte extravasation (19) suggesting a possible novel function of endoglin in the pathophysiology of the vascular system for ENG independent of TGF-β signaling (Figures 1B,C). Several is illustrated by the phenotype of different HHT1 animal lines of evidence support the role of ENG in leukocyte trafficking: models (32). While endoglin heterozygous mice are viable (i) ENG expression is markedly up regulated in endothelial cells and reproduce the vascular phenotype of HHT patients, total of inflamed tissues with an associated inflammatory cell infiltrate loss of ENG expression leads in mice to cardiovascular defects (19); (ii) ENG is up-regulated in the post-ischemic kidney and and embryonic death by mid-gestation (33–35). A conditional ENG-haploinsufficient mice are protected from renal ischemia- knock out mouse model for HHT1 has revealed that the reperfusion injury, due to a reduction of cellular inflammatory arteriovenous malformations (AVMs), induced upon ENG responses (20); (iii) Arterial, venous, and capillary endothelia in loss, appear to be the result of delayed vascular remodeling lymphoid organs are highly reactive with anti-ENG antibodies and inappropriate ECs proliferation responses (36). In ENG and a marked staining pattern is observed in high endothelial null mice, vasculogenesis does not seem affected, suggesting venules (21); and (iv) Although ENG is present throughout that ENG is not required for initial differentiation of ECs or the vascular endothelium, its expression, as compared to veins formation of a primitive vascular plexus. The yolk sac of Eng−/− or arteries, is stronger in capillaries, where most leukocyte embryos has enlarged fragile vessels, while the embryo proper infiltration to organs occurs. shows cardiac cushion defects and delayed maturation of major In addition to leukocytes, other cell types present in the vessels (34). The reason why the total loss of ENG expression circulatory system have also revealed an integrin-mediated leads to cardiovascular defects and embryonic death is not adhesion activity toward endothelial endoglin (22–24) (Figures 1D,E). Thus, endoglin plays a role in integrin-mediated completely understood. Some authors have postulated that adhesion of vascular mural cells to endothelium (23). Also, a the primary defect is in the heart (37), while others suggest new role for endoglin in αIIbβ3 integrin-mediated adhesion of that the loss of smooth muscle cells (SMCs) coverage of the −/− platelets to the endothelium, conferring resistance of adherent endothelium could play a decisive role in the lethality of Eng platelets to detachment has been described (24)(Figure 1E). animals (34).

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FIGURE 1 | Hypothetical model of Endoglin and its role as TGF-β co-receptor and adhesion molecule in different physiological contexts. (A) Canonical pathway that implicates Eng as a co-receptor of TGF-β in ECs. Bone morphogenetic protein 9 (BMP9), and other members of the TGF-β family, bind to an EC receptor complex composed by the type I (R-I) receptor named ALK1 and the type II (R-II) receptor, both exhibiting serine/threonine kinase activity, as well as the auxiliary receptor endoglin. Upon ligand binding, the R-II transphosphorylates ALK1, which subsequently propagates the signal by phosphorylating the receptor-regulated Smad (R-Smad) family of proteins Smad1/5/8. Phosphorylated R-Smads form heteromeric complexes with Smad4, translocating into the nucleus to regulate the transcriptional activity of different target genes. BMP9, Endoglin, ALK1 and Smad4 proteins are encoded by GDF2, ENG, ACVRL1 and MADH4 genes, whose pathogenic mutations give rise to HHT5, HHT1, HHT2, and JPHT, respectively (30). (B) Endothelial endoglin as an adhesion molecule involving its RGD sequence when binding to cell surface integrins from leukocytes, VMCs or platelets. (C) Leukocyte transmigration through the vessel endothelium. Under inflammatory conditions, different soluble factors are released leading to leukocyte adhesion and transmigration through ECs. This process is mediated, at least, by the interaction between leukocyte integrin α5β1 and endothelial endoglin involving the specific recognition of the RGD motif in Eng (22). (D) Adhesion between endothelial cells and vascular mural cells (VMCs). This intercellular association involves, at least, the interaction between integrin α5β1 in VMCs and endothelial endoglin via the specific recognition of the RGD motif in Eng (23). (E) Platelet-dependent hemostasis. Endothelial endoglin via its RGD binds to platelet integrins αIIbβ3 and α5β1, contributing to stabilize platelets adhesion to endothelium (24).

ENDOGLIN INTERACTING PARTNERS IN Ras-related C3 botulinum toxin substrate 2 (Rac-2), involved THE VASCULATURE in endothelial barrier maintenance, cytoskeletal remodeling and cell migration (44). Using in vitro ECs lines derived Previous studies reported that endothelial ENG is associated with from ENG homozygous null, heterozygous, and control mouse several proteins critically involved in endothelial cell adhesion, embryos, Jerkic and Letarte have reported that ENG-deficient proliferation, migration, angiogenesis, and vascular permeability, mouse embryonic ECs are hyper-permeable and unresponsive including integrins (38), zyxin (39), zyxin-related protein 1 to stimulation by VEGF and TGF-β1, factors that regulate (ZRP-1) (40), VEGF receptor type 2 (VEGFR-2) (41), beta- vascular permeability (45). Interestingly, VE-cadherin is barely arrestin 2 (42), and vascular endothelial-cadherin (VE-cadherin) detected in ENG-deficient ECs, a finding that could explain (43). It has been shown that VE-cadherin interacts with several the EC junction destabilization and impairment of TGF- components of the TGF-β receptor complex, including ENG, β signaling. Furthermore, permeability alterations in Eng+/− ALK1, and the TGF-β type II receptor TGFBR2 in adherents mice are in line with findings in patients with Hereditary junctions. Of note, ENG, TGFBR2, and ALK1 also interact Hemorrhagic Telangiectasia type 1 (HHT1). Thus, ECs from with p21-activated kinase (PAK-1), GTP-binding proteins, and HHT1 patients show a disorganized actin cytoskeleton prone to

Frontiers in Medicine | www.frontiersin.org 3 January 2019 | Volume 6 | Article 10 Rossi et al. Endoglin and Endothelial Progenitors cell breaking with changes in shear stress and blood pressure and in vivo and they are involved in blood vessel formation and (46). This reorganization of actin filaments in HHT1 might vascular repair (58, 59). For example, upon vascular injury, tissue lead to vessel hemorrhages and eventual disappearance of the perfusion of blood flow involves not only angiogenic sprouting capillary network, as reported in this disorder. In line with the of ECs from nearby intact vessels, but also the recruitment pathogenic hypothesis of an endothelial dysfunction in HHT1, circulating ECFCs allowing the formation of new blood vessels the involvement of ENG in integrin-mediated trans-endothelial (60). Acting at the interface between blood and tissues, ECFCs leukocyte trafficking was proposed (31). Accordingly, following have a remarkable ability for migration and proliferation, inflammation triggering, the extracellular region of ENG binds to being key cells in angiogenesis and vascular remodeling. It leukocyte integrin α5β1 and promotes leukocyte transmigration is therefore not surprising that perturbations in these critical (31). In contrast, a different experimental approach showed an ECFCs functions contribute to several vascular pathologies. increased gut inflammation and myeloid infiltration in colitis Accordingly, ECFCs have opened a new area for treatment Eng+/− vs. control mice (47). Overall, this novel role of ENG may of cardiovascular diseases using cell therapy, advancing in the contribute to a better understanding of the inflammation process knowledge of vessel reconstruction ability in postnatal life. In and the high incidence of infections in HHT patients (48). this line, much effort has been recently devoted to identify Because pericytes embrace the endothelium to regulate the blood novel molecular targets within ECFCs able to enhance their retinal barrier, the role of endothelial endoglin in this context angiogenic potential. ECFCs are positive for endoglin (CD105), has also been analyzed. Thus, vascular permeability studies VE-cadherin (CD144), CD31, VEGFR2 (KDR), EGFL7, and carried out in vivo showed that retinas of Eng+/− heterozygous CD146, and negative for CD45 and CD14 (58, 59). Among these, mice displayed higher retinal permeability than that of Eng+/+ ENG is emerging as an interesting therapeutic target based on its mice, suggesting a destabilization of the endothelial barrier involvement in angiogenesis and vascular remodeling (46, 61). function due to ENG haploinsufficiency (23). The importance Many strategies of therapeutic revascularization, based on of the interaction between ECs and vascular mural cells is the administration of growth factors or stem/progenitor cells also underlined by studies showing that thalidomide stabilizes from diverse sources including EPCs, have been proposed the vasculature in a mouse model of HHT1 (49). Indeed, and are currently being tested in patients with peripheral pericyte recruitment and stabilization appears as a therapeutic arterial disease or cardiac diseases (62). Some pretreatment of strategy to reduce the severity of epistaxis in HHT (50). Thus, ECFCs with erythropoietin (63), fucoidan (64), soluble CD146 ENG, directly, or as a component of the TGF-β receptor (65), tumor necrosis factor-α (TNF-α) (66), or platelet lysates complex may regulate ECs integrity, whereas its absence may (67, 68) have also been proposed to improve the therapeutic result in vascular hyper-permeability, thus contributing to the efficacy of in vivo administered ECFCs. Interestingly, integrins fetal lethality associated with the homozygous null genotype represent a major molecular determinant of EPCs function. More (39, 40). specifically, integrin α4β1 is a key regulator of EPCs retention Although this review focuses on the role of endoglin in and/or mobilization from the bone marrow, while integrins ECs, it is worth mentioning that endoglin is also expressed α5β1, α6β1, αvβ3, and αvβ5 are involved in EPCs homing, at lower levels in other cell types, some of which present invasion, differentiation and paracrine factor production (69). in the vasculature as vascular SMCs, activated monocytes or The involvement of ECFCs in angiogenesis has been shown to mesenchymal cells (18, 51–53). However, a review on the be essential for ischemic disease recovery (70, 71). Furthermore, endoglin role in these cell types deserves an independent the differentiation of mesenchymal stem cells (MSCs) into mural study. cells appears to be stimulated by their co-culture with ECFCs, whereas overexpression of the Notch ligand Jagged1 in ECs further enhanced the differentiation of MSC into pericytes ENDOGLIN AND ENDOTHELIAL (72). This cooperation prompted some authors to postulate COLONIES FORMING CELLS (ECFCS) an improvement of ECFCs efficiency by co-injecting ECFCs and SMC progenitors (73). Therefore, a combined treatment of The formation of blood vessels from mesoderm is driven by the ECFCs with mesenchymal stem/progenitor cells (MSCs/MPCs) recruitment of undifferentiated cells which can differentiate into appears to operate synergistically, enhancing neovascularization endothelial lineage. In the adult, populations of bone marrow- compared to either individual cell population (74, 75). Recently, derived endothelial progenitor cells (EPCs) are mobilized into we proposed a role for ENG in the ECFCs-MSCs interplay the circulation by stimuli such as estrogen and VEGF (54). involved in the revascularization process (61). Indeed, ENG Interestingly, human EPCs could derive from very small silencing in ECFCs markedly inhibited ECFCs adhesion to MSCs embryonic like stem cells (55) that can also give rise to in vitro, without affecting MSCs differentiation into perivascular hematopoietic cells (56). Two populations of EPCs have cells or other mesenchymal lineages. Mesenchymal stem cells been differentially characterized from circulating endothelial (MSCs) increase muscle recovery of ECFC in HLI model and progenitors: (i) early EPCs that exhibit a hematopoietic profile we found that ENG-silenced ECFCs co-injected with MSCs in with a genomic fingerprint similar to monocytes; and (ii) ECFCs, mice abolish beneficial effects of MSCs leading to decreased vessel also known as blood outgrowth endothelial cells (BOECs), which density and foot perfusion upon ischemia. Our results suggest are considered late EPCs with an endothelial-like genomic profile ENG involvement in the crosstalk between ECFCs and MSCs, (57). ECFCs display an intrinsic tube forming capacity in vitro leading to vessel formation and stabilization (51)(Figures 2A,B).

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FIGURE 2 | Endoglin in ECFCs. (A) Role of endoglin matrigel vascularization in vivo. Matrigel plugs were mixed with either ECFCs treated with control siRNA plus mesenchymal stem cells (MSCs) (left) or with ECFCs treated with endoglin speciﬁc siRNA plus MSCs (right). The Matrigel mixture was injected into nude mice and the number of vessels was analyzed after 1 week. Control plugs display a marked vascularization with functional vessels (presence of erythrocytes). Plugs with endoglin-silenced ECFCs display less vascularization than controls, suggesting an important role for this protein in cell adhesion (23). (B) MSCs combined with ECFCs, accelerate muscle recovery in a mouse model of hind limb ischemia, through an endoglin-dependent mechanism. After femoral ligature and retro-orbital injection of ECFC+MSC Doppler analysis shows a revascularization induced by co-injection of ECFCs plus MSCs (left). This synergistic effect is abolished when endoglin is previously silenced in ECFCs (right) (61). (C) Mechanisms involved in the synergy between ECFCs and MSCs. (i) Mutual paracrine effect between ECFCs and MSCs involving growth factors like VEGF and PDGFBB (76, 77). (ii) ECFCs-induced differentiation of MSCs into perivascular cells via Notch-Jagged1 (72) (iii) Adhesion between ECFCs and MSCs involving endoglin (61).

As postulated in Figure 2C, three steps may be involved Thus, the ENG cytoplasmic domain binds to zyxin and ZRP1, during the formation of stable vessels by ECFC and MSC which concentrate at focal adhesions within actin polymerization and/or perivascular cells: (i) Mutual paracrine effects by VEGF points. Consequently, the decrease of ENG levels in HHT1 and/or PDGF-BB (76, 77). (ii) ECFCs-driven perivascular ECFCs could be related to the cytoskeleton alteration. maturation of MSC and/or SMC progenitors via the notch- dependent pathway; and (iii) Adhesion between ECFCs CONCLUSION and perivascular cells in an ENG-dependent manner. The relevance of endothelial ENG adhesive properties in Blood vessel formation and remodeling are essential processes mature vessel formation in mice (61) is in agreement with in the maintenance of tissue homeostasis and function, a previous report on the active role of human ENG in the and therefore, their alteration causes a variety of pathologic adhesion between mature ECs and vascular mural cells conditions. ENG involvement in the function of mature and (23). progenitor endothelial cells is a hot topic not completely Relevance of ENG in ECFC has been demonstrated also understood and developed yet. Recent findings underline in ECFCs isolated directly from HHT1 patients. Indeed, these the importance of ENG not only as a co-receptor for several ECFCs have disorganized and depolymerized actin fibers and ligands of the TGF-β family, but also as a molecule involved impaired tube formation, as compared to healthy ECFCs (46). in cell adhesion, which can regulate ECFCs behavior in The molecular basis for the abnormal behavior of HHT1 ECFCs the context of angiogenic processes. It is recognized that appears to be mediated, at least in part, by the regulatory in mature ECs, ENG plays a key role in angiogenesis and role of endoglin and we can speculate via its interaction blood vessel homeostasis, becoming a potential therapeutic with zyxin family members involved in the actin cytoskeletal target for pro- and anti-angiogenic approaches in the organization as it was proposed for endothelial cells (39, 40). treatment of diseases such as HHT, cancer, preeclampsia,

Frontiers in Medicine | www.frontiersin.org 5 January 2019 | Volume 6 | Article 10 Rossi et al. Endoglin and Endothelial Progenitors diabetes complications or post-ischemic disease. While ENG and platelets, independently or in parallel to its TGF-β co- expression is relatively low in quiescent ECs, it is upregulated receptor role. in the active endothelium involved in angiogenesis and While evidence-based therapeutics are coming into play in vessel remodeling and permeability. In these processes, the traditionally empirical base for endoglin related protein endothelial ENG regulates ECs proliferation, migration, and derivatives, future studies of ENG in endothelial progenitor cells actin cytoskeletal organization, leukocyte extravasation, and may pave the way for a better understanding of its function in the interaction with mural cells. In addition, a role for ENG in vascular system and for the development and understanding of platelet-endothelium interaction, including the resistance of the use of these immature cells as a cell therapy product. adherent platelets to shear under inﬂammatory conditions, was also recently proposed, opening new perspectives on ENG AUTHOR CONTRIBUTIONS functions. This review highlights the emerging roles of endothelial ER wrote the manuscript. CB provided helpful suggestions and ENG as a cell adhesion molecule in mature and progenitor contributed to writing the manuscript. DS wrote the endothelial cells interacting with vascular mural cells, leukocytes, manuscript and provided funding support.

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